Phosphorylation of dihydroriboflavin



Patented July 22, 1952 RIBOFLAVIN Leo A. Flexser, Upper Montclairjami Walter G. I 57 Farkas, 'Nutl'ey, N. J., assignors' to Hofimann-- La Roche Inc., Nutley,

New Jersey N. "L a corporationoi 1 f No Drawing; App ication July 15, 1950,

II Se'rial'No.1'74, 1g1 I -Claims. 3 (or. 260-2113) This invention relates to anovel method for preparing the 'monophosphoric acid ester of dihydro-riboflavin. This ester can, be readily oxidized to the useful and physiologically important monophosphoric acid ester of riboflavin. Accordingly, our invention also embraces the steps of phosphorylating dihydro-riboflavin to form'the monophosphoric acid ester of dihydroriboflavin and oxidizing'the latter to form the monophosphoric acid ester of riboflavin. 1

Our invention offers the additional advantage that it employs the readily accessible dihydroriboflavin as a starting material. Dihydroriboflavinis, more readily obtained than riboflavin itself from dilute aqueous solutions since it is much less soluble and is, therefore, more easily isolated than riboflavin from fermentation process liquors or other dilute solutions.

Inour copending application Ser. No. 141,585, filed'January 31, 1950, now abandoned, we disclose that riboflavin may be phosphorylated by chlorophosphoric acids toyield various products which comprise mono(monochlorophosphoric acid) salts and mono(dichlorophosphoric acid) salts of riboflavin monoesters of phosphoric acid, polyphosphoricacids, chlorine-containing phosphoric acid, and chlorine-containing polyphosphoric acids. These-phosphorylated riboflavin compounds may all be hydrolyzedto the monophosphoric acid ester of riboflavin. The new method disclosed in our aforementioned application for phosphorylating riboflavin consists of pretreating phosphorus oxychloride (POCls) with water to form a chlorophosphoric acid in which riboflavin is soluble and whereby'it is readily phosphorylated at room temperature. The .chlorophosphoric acids are readily prepared by reacting phosphorus oxychloride with about 1 to 2 moles of water. The treatment of phosphorus oxychloride with water follows the course;

We have now discovered that dihydro-riboflavin, in spite of' its having a lower solubility than riboflavin, may also be phosphorylated by the process disclosed for riboflavin in the aforesaid copending application Ser. No. 141,585. The resultant products may also be hydrolyzed to the monopho'sphoric acid ester of dihydro-riboflavin, which can in turn be oxidized to produce the monophosphoric acid esterof riboflavin.

Preferably, the reaction is carried out with an excess of phosphorylating agent and for a sufficient length of time to insure that no chlorophosphorylated depending dihydro-riboflavin' remains unphosphorylated. The products which are thereupon obtained contain atleast two atoms 'of phosphorus and at leastone chlorine atom. The extent of the polyphosphorylation depends upon the length of time the dihydro-riboflavin is allowed to react with the water-pretreated P0013; In the caseof P0013 pretreated with one mole of water, the reactions may be formulated thus:

ROH ZHOPOCI: [RQEPH].HOPOC12( wherein ROI-I represents dihydro-riboflavin.

The above monochlorophosphorylated dihydroriboflavin chlorophosphate may then be further upon the reaction time'asfollows: Y

, I O I (a) 110F001, [RoPoPoHJJioPoOh c1 c1 (b) and/or 0 0 (c) ROPOPCI .HoPooh Compounds (b) and (0) may be 'further phosphorylated.

In the case of POCla pretreated with 2 moles of water the corresponding reactions are:

Upon further treatment with the 2Hz0 pretreated PO C13, the following occurs:

- taining phosphoric acid andchlorine-containing polyphosphoric acids; c I

Hydrolysis of any of the intermediates pro duced with the chlorophosphoric acids results first in the splitting off of the salt-forming HOPOClzor (HO) zPOCl, as the case may. be,

followed by liberation of .dihydro-riboflavin- I 3 portion of dihydro-riboflavin (2), (3), or (4'), monophosphoric acid ester may also be formed. It is to be understood that we do not wish to limit ourselves by the above theoretical interpre- V tation of the formation of our new dihydro riboflavin. compounds containing phosphorus'and chlorine, but rather intend it as a reasonable explanation of the reaction which may be in-' volved. The reactions given herein set forth a synthesis with postulated. structures 'forjthe' intermediates involved.

We claim:

1. A process of phosphorylating dihydro-ribofiaVin which comprises reacting dihydro-riboflavin with a member selected from the group consisting-.ofthe two acids representedmy-the formulae (HO) 2POC1 andHOBO-Clz.

2. A process of phosphorylating dihydro-ribo- "flavin which comprises reacting dihydro-ribothat although the outline given above is -based uponwhat we consider to be the most probablecourse of reaction, we do not wish to limit our invention to any specific theory of reaction or structure of products.

While the aforementioned deltas-absce s ester salts may be isolated for purposes of purifiviii. with the acid HOP- 012.

fi avin with the acid represented by the formula 'lO It will be understood (HOMPOCL 3. A process of phosphorylating dihydro-riboliavin" which comprises reacting dihydro-riboflarepresented. by the formula 4. A processof phosphorylating dihydro-ribo- Mflavin which comprises reacting dihydro-riboflawith a member selected from the group concation or for preparation of the derivatives therof '(for instance for reaction with ammonia), it iSiI IQLiFTIIBCBSSaIYLJIO efiect such isolation for the preparation: of;- ;the riboflavin monophosphoric acid :esterg 'lfn thelatterginstance, the. reaction phoric acid ester and the new'inte'rmediate products. It is intended that this example be illustrativeoflthe" process'and not restrictive-of the scope of our invention.

Example To'61L3. grams (0.4 mole) of phosphorus oxychloride were added slowly, with good agitation and-with cooling to :about room' temperature, 7 .2 grams (0.4 mole) of water. The mixture was stirred until;evolution. of: hydrogen chloride gas appeared mostlyrfinished and allowed 'tostand overnight (about16 hours). Then 3.78 grams of dihydro riboflavin were added withstirring. S0- lution occurred after a short time and H01 was evolved-; '-The-solution vwas stirred-for 5 hours; at

'room temperature.- --It was then;poured-into 60 0 vcc. of water, To the solution of dihyclro-ribofla-Vin--monophosphoric-acid ester thus obtained was added a quantity of hydrogen peroxide sufficientto cause oxidation as evidencedby the color change to typicalye'llow and the appearance .of typicalfluorescence" of riboflavin mcnophosphoric acid ester. Upon standing, riboflavin rnonophos-- phoric jacidester crystallized. The 'jcompounfd meltedjatj about. 195 C.,, and analyzed correctly filehof this patent: is 4 TEDsTATEs PATENTS '1' Date forcthel'iiloi dp iQsi hcric acid esterof jriboflavin corresponding to the formul s,cnnlpmoseosnz. The optipal rotation of a two, per centsolution in nce't ated hydrochloric acid was tentioinet'ric titration with sodium" hydrox well'defined point of inflection in' the titratip 'curvejwas obtained at pH 4.5 uponth'e addi ti'gn of 'exacflyone equivalent oi alkali. 'A's'e'condfinfle'ction point at pH B;.5 was obtained upon on of'a second equivalentoi alkali.

sistingof the two acids represented by the formulae (HO) zPOCl and I-IOBOC12, and hydrolyzing the reaction product to produce dihydroriboflavin-"5' -monophosphoric acid ester.;

5.1A'1proc'ess of phosphorylatings dihydro-rribo fiavin which comprises reacting dihydro-ribofl'avin with the acid: represented by the formula (HOizPoClpand hydroly'zing the reaction ,prod

not to produce dihydro riboflavin-ii'-monophosphoric acid ester. 7 7 6"'A process of phosphorylating dihydro-ribo fiavin'which comprises reacting dihydroeribofla- Vin with the acid "represented by the formula HOFO'Clz, andQhydrOlyZingZtIie reaction product to'pr'o'du'ce'dihydro-riboflavin-5' -monophosphoric acid-ester; 'x 1 "7. A' process of 'phosphorylatingvdihydro-rihm fi'avin which comprises lreacting tdihydroqibofi'a-vin with'a member selected from the'group consisting of the two acids represented bylthe formulae (HO) {P001 and HOPOCl'z, hydrolyzing the reaction product to produce 'dihydro ribofiavin-5' -inoiiopliosphoric acid ester, and oxidi'z;

ing the dihydro' ribofiavin} 5 1'n'ono'phosphoric acid ester-to produce riboflavin-"5': i monop-hos phi ricacid ester. p v r s 8. 'A chlorine-containingproduct produced 'according to the process ofclaim 1. i 9; A'ch1orineconta'ining product produced according to the proces'sof claim 2. 1 1 I1 10: chlorine containing product produced ancordin gito the proces' s'ofclaim 3';

WA TER FARKAS;

REFERENCES crren ,Iilhe iollowingljreferences are'of recordinthe Number Name "2.0512,?029 "Harris' Aug. 25, 1936 2,111,491 Kuhn Mar. 15, 1938 2,224,695 2,256,380 I 2,394,829 7 Whitehill et 2.1.' 1 810.12, 1946 "2,490,573 Atherton Dec. 6, 1949 --2,53 5,385 Bewogel Dec. 26,1959,

' FOREIGN PATENTS; Number; .Country Date. I 404,684;

GreatlB ritalin $1 .11;" an. "LO'ITHERV'REFERENCES w 3 Chem. Ab stQ, vol. 39 (1945)., page 2734;

J. Chem. Soc. 1945),pages 106-12 i citing 

1. A PROCESS OF PHOSPHORYLATING DIHYDRO-RIBOFLAVIN WHICH COMPRISES REACTING DIHYDRO-RIBOFLAVIN WITH A MEMBER SELECTED FROM THE GROUP CONSISTING OF THE TWO ACIDS REPRESENTED BY THE FORMULAE (HO)2POCL AND HOPOCL2. 